1. Field of the Invention
The present invention relates generally to the fields of medical imaging, analysis, monitoring and diagnostics. More particularly, it provides apparatuses and methods for analyzing proteins in samples. Even more particularly, it may be used in the direct profiling of diseased tissue by mass spectrometry; this, in turn, may be used for the assessment of disease classification, development and treatment.
2. Description of Related Art
Every year about 1.5 million Americans are diagnosed with cancer. Tragically about 500,000 people die of the disease every year. Cancer can affect people in a variety of different ways: about 11% of cancer patients are diagnosed with colorectal cancer, 15% with prostate cancer, 20% with lung cancer, 15% with breast cancer, and 2% with brain cancer. The ability to effectively identify specific tumor markers in proliferating areas of the tumors would therefore be a beneficial step in diagnosing, monitoring, analyzing, and treating such tumors and, in general, a wide variety of other ailments.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is an analytical technique having high sensitivity, ease of use, and compatibility as an effective off-line method for different types of sample analysis. Static sampling systems using MALDI MS have demonstrated extremely high sensitivities, as illustrated by attomole sensitivity for the analysis of peptides contained in complex physiological salt solutions. Further, matrix precoated cellulose targets have been used to analyze 100% aqueous samples without the need of further treatment with organic solvents.
Several reports have described the use of MALDI for the analysis of specific peptides in whole cells. Several papers describe the analysis of some neuropeptides directly in single neurons of the mollusk Lymnaea stagnalis. Isolated neurons were ruptured, mixed with small volumes of matrix, and analyzed. The ability of MALDI MS to be used to elucidate some of the metabolic processing involved in neuropeptide production from precursor peptides has also been demonstrated. Also, a single neuron from Aplypsia californica was analyzed for several specific neuropeptides using a procedure involving removal of excess salt by rinsing with matrix solution.
A considerable amount of work has been described for use of secondary ion mass spectrometry (SIMS) for the spatial arrangement of elements in surfaces of samples including biological tissue and organic polymers. In addition, there have been recent efforts to apply the SIMS technique to organic compounds and metabolites in biological samples. At least one report describes conditions for generating secondary ion mass spectra from samples with choline chloride and acetylcholine chloride deposited onto specimens of porcine brain tissue. Samples were then exposed to a primary ion beam of massive glycerol clusters. Images generated from the spacially arranged SIMS spectra were obtained that reflected the identity and location of the spiked analytes.
U.S. Pat. No. 5,808,300 which is incorporated herein by reference, describes a method and apparatus for imaging biological samples with MALDI MS. Its techniques can be used to generate images of samples in one or more m/z pictures, providing the capability for mapping the concentrations of specific molecules in X, Y coordinates of the original biological sample. Analysis of a biological sample can be carried out directly or to an imprint of the sample. The image attained in the analysis can be displayed in individual m/z values as a selected ion image, as summed ion images, or as a total ion image. The imaging process may also be applied to other separation techniques where a physical track or other X, Y deposition process is utilized.
U.S. Pat. No. 5,272,338 which is incorporated herein by reference, describes a specific instrument setup using a liquid metal ion source to ionize a sample in a mass spectrometer, and then a laser beam to irradiate the ejected molecules and resonantly ionize them. The method involves a technique commonly referred to as SIMS/cross beam laser ionization.
U.S. Pat. Nos. 5,372,719 and 5,453,199 which are incorporated herein by reference, disclose techniques for preparing a chemically active surface so that when a sample is exposed to this surface, a chemical image of the sample is deposited on the surface. The disclosed methods involve the separation of molecules by sorbents.
U.S. Pat. No 5,607,859 which is incorporated herein by reference, describes a method for the MS determination of highly polyionic analytes by the interaction of oppositely charged molecules.
U.S. Pat. No. 5,569,915 which is incorporated herein by reference, discloses an MS instrument for fragmenting molecules in the gas phase.
U.S. Pat. No. 5,241,569 which is incorporated herein by reference, describes neutron activation analysis for detecting gamma rays and beta-electrons from radioactively labeled samples. This technique may be used to locate elements in a sample.
Although techniques referenced above show at least a degree of utility in performing certain types of general analysis, shortcomings remain. For instance, current techniques used for rapid decisions in the clinical laboratory often involve the analysis of frozen sections using light microscopy. Such techniques, however, are, at times, non-specific and inaccurate. More importantly, conventional technology does not involve techniques that effectively employ the capability of MALDI MS to analyze and effectively depict a quantity of molecules of interest with a specific atomic mass or within a selected atomic mass window or as a function of their position on the test sample. Further, conventional technology does not employ MALDI MS techniques to effectively analyze proteins in clinical samples. Still further, conventional technology does not use MALDI MS techniques to perform direct profiling of diseased tissue, which in turn may be used for the assessment of disease classification, development and treatment.
The molecular analysis and imaging of proteins and other biological materials in tissues is an important part of medical imaging, monitoring, analysis, diagnostics, disease classification, and treatment of a variety of disorders. As used herein xe2x80x9cbiological materialxe2x80x9d is to be interpreted broadly and can include, for example, nucleic acids, lipids, carbohydrates or any molecule covered in Biochemistry (Stryer, et al., 2002). Specifically, molecular analysis and imaging may be used as an integral building block in strategies designed to locate specific proteins that are more highly expressed in tumors relative to normal tissue. Likewise, it may be used to locate specific proteins diminished in expression relative to normal tissue. One of the many aims of the techniques of this disclosure is the molecular analysis and imaging of peptides and proteins in brain tumors, specifically in human glioblastoma.
Generally speaking, this disclosure involves mass spectrometric techniques of matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) to image and detect proteins and other biological materials in tissue. Certain aspects of this disclosure employ the molecular specificity and sensitivity of mass spectrometry (MS) for the direct mapping and imaging of biomolecules present in tissue sections. This technology has been developed using MALDI MS and may be used for the analysis of peptides and proteins present on or near the surface of samples, such as tissue sections. Imaging MS brings a new tool to bear on the problem of unraveling and understanding the molecular complexities of cells. It joins techniques such as immunochemistry and fluorescence microscopy for the study of the spatial arrangement of molecules within biological tissues.
In one respect, the invention is a method of analyzing proteins within a sample. A specimen including an energy absorbent matrix is generated. The specimen is struck with a laser beam such that a predetermined first laser spot on the specimen releases first sample proteins. The atomic mass of the released first sample proteins is measured over a range of atomic masses. The specimen is moved relative to the laser beam a predetermined linear distance functionally related to a size of the predetermined first laser spot. The specimen is struck again with the laser beam such that a predetermined second laser spot on the specimen releases second sample proteins. The atomic mass of the released second sample proteins is measured over a range of atomic masses. An atomic mass window of interest within the range of atomic masses is analyzed to determine the specific proteins within the sample. The determined specific proteins can be mapped as a function of the spatial arrangement.
In other respects, the specimen may include tumor-bearing tissue. The tumor-bearing tissue may include brain tissue. It may include prostate tissue. It may include colon tissue. The step of generating specimen may include generating a fresh, or frozen section. It may also include generating individual cells or clusters isolated by laser-capture microdissection or other cell isolation techniques. As used herein xe2x80x9cseparate layersxe2x80x9d refers to separate two dimensional sections of a tumor. The step of analyzing the atomic mass window of interest may include graphically depicting the mass of proteins within the atomic mass window of interest as a function of the linear distance between the first spot and the second spot. The specimen may be dried prior to being struck with laser beams. Proteins within the atomic window of interest from the first laser spot may be analyzed while the laser beam strikes the second laser spot. The linear distance of movement between successive laser spots may be less than twice the width of each of the successive laser spots. The step of identifying the specific proteins may involve extraction, HPLC fractionation, proteolysis, mass spectrometric sequencing of one or more fragments and protein database searching.
In another respect, the invention is a method for classifying disease. A diseased specimen including an energy absorbent matrix is generated. The specimen is struck with a laser beam such that a predetermined first laser spot on the specimen releases first sample proteins. The atomic mass of the released first sample proteins is measured over a range of atomic masses. The specimen is moved relative to the laser beam a predetermined linear distance functionally related to a size of the predetermined first laser spot. The specimen is again struck with the laser beam such that a predetermined second laser spot on the specimen releases second sample proteins. The atomic mass of the released second sample proteins is measured over a range of atomic masses. An atomic mass window of interest within the range of atomic masses is analyzed to determine the spatial arrangement of specific proteins within the sample. The specific proteins are identified as a function of the spatial arrangement, and the identified specific proteins are correlated with one or more diseases to classify the diseased specimen.
In another respect, the invention is a method for monitoring the development of a specimen over time. A specimen including an energy absorbent matrix is generated at a first time. The specimen is struck with a laser beam such that a predetermined first laser spot on the specimen releases first sample proteins. The atomic mass of the released first sample proteins is measured over a range of atomic masses. The specimen is moved relative to the laser beam a predetermined linear distance functionally related to a size of the predetermined first laser spot. The specimen is again struck with the laser beam such that a predetermined second laser spot on the specimen releases second sample proteins. The atomic mass of the released second sample proteins is measured over a range of atomic masses. An atomic mass window of interest within the range of atomic masses is analyzed to determine the spatial arrangement of specific proteins within the sample. The specific proteins are identified as a function of the spatial arrangement. These steps are repeated, and the specific proteins identified at the first time are compared with the specific proteins identified at the second and subsequent times to monitor the development of the sample or, more particularly, disease of the sample.
In other respects, the method may also include correlating the identified specific proteins and other biological materials at the first or second times with one or more diseases. The method may also include treating those one or more diseases.
In another respect, the invention is an apparatus for analyzing a sample containing proteins. The apparatus includes a laser source, a moving mechanism, a mass analyzer, a computer, a display, and means for correlating atomic mass. The laser source is adapted for sequentially striking a specimen with a laser beam at a plurality of laser spots on the specimen for sequentially releasing sample proteins from each laser spot. The moving mechanism is adapted for sequentially moving the specimen relative to the laser beam a predetermined linear distance functionally related to the size of the laser spots prior and subsequent to the movement. The mass analyzer is adapted for measuring the atomic mass of the released sample proteins over a range of atomic masses. The computer is adapted for receiving atomic mass data from the mass analyzer. The display is adapted for depicting atomic mass within an atomic mass window of interest as a function of individual laser spots on the specimen. The means for correlating atomic mass includes means for performing such a correlation so that atomic masses may be correlated with one or more specific proteins. The means for performing this function are described herein and in the incorporated references and may include a wide variety of mechanisms including databases or any other computerized methodology and software for implementing the correlation steps discussed herein.